Rebaudioside A (Reb A) is a new natural sweetener extracted from Stevia rebaudiana with intense sweetness, low calorific content and good stability, etc. Compared to the other main components of steviol glycosides, rebaudioside A (RebA) has the highest sweetness. The sweetness of rebaudioside A is at least 450 folds stronger than sucrose, but its calorific content is 300 folds less than sucrose. Besides, rebaudioside A has intense sweetness, white color, pure sweet taste and no peculiar smell. Therefore, it is the best nature alternative for sucrose and the chemically synthetic sweeteners, and it is called as “the third-generation sugar source” in the world.
FIG. 1 shows the structures of a portion of steviol glycosides extracted from Stevia rebaudiana, which has different side chain modifications due to their different R1 and R2 (see Table 1). Stevia sugar will have purer sweet taste if it contains more rebaudioside A and thus it will be accepted by more consumers. Therefore, the content of rebaudioside A in the product must be enhanced during production of stevia sugar.
TABLE 1Steviol glycosides extracted from Stevia rebaudianaNo.CompoundR1R21SteviolHH2SteviolmonosideHβ-Glc3SteviolbiosideHβ-Glc-β-Glc(2→1)4Rubusosideβ-Glcβ-Glc5Steviosideβ-Glcβ-Glc-β-Glc(2→1)6Rebaudioside Aβ-Glcβ-Glc-β-Glc(2→1)β-Glc (3→1)7Rebaudioside BHβ-Glc-β-Glc(2→1)β-Glc (3→1)8Rebaudioside Cβ-Glcβ-Glc-α-Rha(2→1)β-Glc(3→1)9Rebaudioside Dβ-Glc-β-Glc(2→1)β-Glc-β-Glc(2→1)β-Glc (3→1)10Rebaudioside Eβ-Glc-β-Glc(2→1)β-Glc-β-Glc(2→1)11Rebaudioside Fβ-Glcβ-Glc-α-Xly(2→1)β-Glc(3→1)12Dulcoside Aβ-Glcβ-Glc-α-Rha(2→1)
Currently, steviol glycosides have been widely used in food, beverage, medicine and cosmetic industries. The recent studies show that steviol glycosides can be used to prevent hypertension, diabetes and heart disease, etc. Therefore, there is a rapid need for steviol glycosides in the recent years.
Commercially available rebaudioside A currently is mainly extracted from the leaves of Stevia rebaudiana. The preparation process mainly includes the steps of drying and grinding the leaves of Stevia rebaudiana, extracting in a liquid phase, removing impurities, treating by resin, drying by spray and refining, etc. Generally, the leaves of Stevia rebaudiana may contain up to 4% to 20% of stevia sugar calculated by dried weight. However, there are many problems, including a great quantity of lands required for planting Stevia rebaudiana, different quantity of Stevia rebaudiana used for production of stevia sugar, low conversion efficiency of the raw material and low purity of the extracted product. Therefore, it is necessary to develop a production method for safely producing rebaudioside A in a large scale, with the raw material being readily obtained and the extraction method being simple.
As the development of techniques in synthetic biology during the last ten years, it is possible to produce various heterologous compounds by microorganisms. This synthesis has advantages of low cost, small production area and easy control of product quantity. However, synthesis of rebaudioside A by heterologous organisms is not reported to date. The key technical difficulty is that the glycosyltransferase used for converting steviolmonoside to steviolbioside was not known. Therefore, there is an urgent need to overcome the existing technical difficulty to achieve microbial synthesis of rebaudioside A.